Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;... – Culture – maintenance – or preservation techniques – per se
Reexamination Certificate
2000-05-19
2004-01-27
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Plant cell or cell line, per se ; composition thereof;...
Culture, maintenance, or preservation techniques, per se
C435S420000, C435S431000, C435S001100, C435S001300, C436S018000
Reexamination Certificate
active
06682931
ABSTRACT:
FIELD OF INVENTION
This invention relates to a method for improving the growth and regeneration potential of embryogenic cell and tissue cultures of coniferous plants retrieved from cryopreservation. In particular, this invention relates to the use of abscisic acid in the post-cryopreservation recovery medium to improve both the growth and somatic embryo production of embryogenic cell and tissue cultures of conifers, thereby enabling more rapid proliferation of the embryogenic cultures and a subsequent increase in the yield of somatic embryos. This method is well-suited for employment with a number of biotechnological uses of embryogenic cultures of coniferous plants retrieved from cryopreservation, including use with embryogenic cultures of coniferous plants and with genetically transformed embryogenic cultures of coniferous plants for producing clonal planting stock useful for reforestation.
BACKGROUND OF THE INVENTION
Cryopreservation is the storage of living cells at ultra-low (cryogenic) temperatures, usually in liquid nitrogen (−196° C.) or in its vapor phase (about −150° C.). Cryopreservation is the preferred method for long-term storage and “banking” of valuable in vitro biological material used in or derived from biotechnology. At cryogenic temperatures the biological activity of the cells and tissues is halted. The cells and tissues remain viable throughout the cryopreservation process due to the application of various cryoprotective procedures (Benson et al. 1998).
There are several methods of freezing used in cryopreservation of biological materials such as living cells or tissues. The basic method is to rapidly cool the biological material or to directly plunge the material into liquid nitrogen. However, this method only works on tissues which remain viable at low moisture content levels. For example, many temperate zone seeds (such as pine seeds which have been dried to below about 15% water content) can be successfully cryopreserved using this method.
A different cryopreservation method is frequently used where the biological material has a relatively high moisture content or where the material may not tolerate dehydration to a lower moisture level. This method involves first treating the biological material with a cryoprotective chemical or combination of cryoprotective chemicals. The treated material is subsequently cooled to about −40° C., then rapidly cooled (e.g., directly plunged into liquid nitrogen) to cryogenic temperatures. This method is the one most frequently employed for cryopreservation of in vitro derived cells and tissues.
Another well-known cryoprotective method is “vitrification.” This method involves the treatment of biological materials with high levels of cryoprotective chemicals in combination with a rapid cooling of the treated materials to cryogenic temperature.
Regardless of the cryogenic freezing method employed, recovery of viable cells after cryopreservation is dependent upon both pre-cryopreservation and post-cryopreservation treatments. In vitro manipulation of the plant tissues or cells in the second and third cryogenic freezing methods noted above are essential to most pre-cryopreservation and post-cryopreservation recovery protocols (Benson et al. 1998). Pre-cryopreservation treatments commonly include the application of a cryoprotective chemical such as glycerol or dimethyl sulfoxide (DMSO). Also, the osmotic potential of the in vitro culture medium is often decreased in the pre-cryopreservation treatment of tissues or cells via the addition of sugars or sugar alcohols such as sucrose, sorbitol, and the like. Post-cryopreservation treatments commonly include the dilution of the cryoprotective chemicals and the osmoticants in the culture medium. Such traditional pre-cryopreservation and post-cryopreservation procedures are commonly practiced and are familiar to those skilled in the art of cryopreservation of plant tissues and cells.
Culture media in which the tissues or cells are grown and proliferated during both the pre-cryopreservation and post-cryopreservation phases typically contain six groups of ingredients: inorganic nutrients, vitamins, organic supplements, a carbon source (i.e., sugars), phytohormones (e.g., auxins or auxins and cytokinins), and a gelling agent for semi-solid or gelled medium. Thus, a commonly used pre-cryopreservation medium for embryogenic cultures would include a standard culture medium (e.g., a medium containing inorganic nutrients, vitamins, organic supplements, and sucrose like that taught by Murashige and Skoog (1962) or a modification thereof) coupled with an auxin, possibly a cytokinin, sorbitol, and DMSO. A typical post-cryopreservation medium for embryogenic cultures would include a standard culture medium, an auxin and possibly a cytokinin, but would be devoid of the cryoprotective chemicals and additional osmotic agents. Frequently, the tissues and cells are initially cultured during post-cryopreservation for a very brief period (typically, one day) on a temporary recovery medium to allow both the cryoprotective chemicals and the additional osmotic agents used in the pre-cryopreservation medium to diffuse out of the tissue and cells. The tissues or cells are then transferred to the same (fresh) medium, lacking cryoprotective chemicals and osmotic agents, to induce recovery and growth. The actively growing tissues or cells can then be utilized for regeneration of plants, or for other biotechnological and genetic engineering purposes.
A significant problem facing those who work with cells and tissue cultures from trees is how to rapidly recover and multiply by proliferation the embryogenic cultures during the post-cryopreservation phase of the somatic embryogenesis process. Somatic embryogenic cultures are employed in the regeneration of trees for clonal propagation, and in the genetic transformation and subsequent regeneration of transgenic trees. It is well-known that embryogenic cultures in general, and pine embryogenic cultures in particular, decline in regeneration potential as the time in culture increases. It is, therefore, important to decrease the length of time taken to multiply or bulk-up the cultures for use in clonal propagation or genetic transformation. It is also believed that increased time in culture may increase the probability of deleterious genetic changes or mutations that result in unwanted somaclonal variation. Such variations are particularly undesirable in clonal propagation and genetic engineering processes. Thus, a central problem or challenge in somatic embryogenesis systems is the need to keep the time in culture to a minimum, while simultaneously producing large amounts of embryogenic tissue or cells which have the potential to produce large numbers of harvestable somatic embryos. This invention addresses the restraints imposed on such systems due to slow growth and recovery during a specific step of the process, namely, the post-cryopreservation recovery phase.
Propagation by somatic embryogenesis refers to methods whereby embryos are produced in vitro from embryogenic cultures. The embryos are referred to as somatic because they are derived from the somatic (vegetative) tissue, rather than from the sexual process. Vegetative propagation via somatic embryogenesis has the capability to capture all genetic gain of highly desirable genotypes. Furthermore, these methods are readily amenable to automation and mechanization. These qualities endow somatic embryogenesis processes with the potential to produce large numbers of individual clones for reforestation purposes.
It was not until 1985 that somatic embryogenesis was discovered in conifers (Hakman et al. 1985) and the first viable plantlets were regenerated from conifer somatic embryos (Hakman and von Arnold 1985). Since 1985, conifer tissue culture workers throughout the world have pursued the development of somatic embryogenesis systems capable of regenerating plants. The goal of much of this work is to develop conifer somatic embryogenesis as an efficient propagation system for producing cl
Becwar Michael Ryan
Krueger Sharon Anne
Boabinski Thomas A.
Lankford , Jr. Leon B.
McDaniel Terry B.
MeadWestvaco Corporation
Reece IV Daniel B.
LandOfFree
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